摘要
早产胎膜早破(preterm premature rupture of membrance,PPROM)是引起早产的主要原因之一,占到早产的25%,约30-40%的早产与PPROM有关。其中在妊娠36周前的PPROM会出现妊娠期较为严重的并发症,其发病率近年来有增加趋势。早产胎膜早破作为占早产原因1／3的产科并发症,早预测早预防具有重大临床意义,能一定程度降低早产率,提高孕产妇及新生儿生存率。
长期以来,胎膜早破是早产临产前的一种危机,早产常与胎膜早破合并存在,胎膜早破使早产成为不可避免,两者互为因果。因此,对PPROM高危因素的分析对其早预防早诊治显得尤为重要,具有一定临床意义。国内外大量研究显示基质金属蛋白酶(MMPs)是一族能降解细胞外基质中所有蛋白成分的内源性酶,对细胞外基质中相应成分的降解,可调控细胞外基质含量和胎膜结构,引起胎膜组织结构的弱化,从而导致胎膜早破发生。随着研究深入,对于早产胎膜早破的病因学研究已进入基因水平。研究发现由于母体与胎儿之间的MMPs基因多态性现象与PPROM有关,导致存在易感基因的孕妇发生PPROM的风险也就增加。易感基因的特点是基因的变异本身并不直接导致疾病的发生,而只造成机体患病的潜在危险性增加,一旦外界有害因素介入,即可导致疾病发生。不同群体和个体对疾病的易感性、抵抗性以及其他生物学性状有差别,其遗传学基础是人类基因组DNA序列的变异性,其中最常见的是SNP。近期有国外学者研究发现胎儿MMP家族的遗传变异中MMP-1、MMP-9、MMP-8转录启动子区域的某些基因多态性(single nucleotide polymorphism, SNP)与PPROM有关。
但是,目前对于与PPROM发病机制相关的MMPs SNP筛查,以及其基因多态性结合母体因素共同研究分析其发病原因的研究不多,且仅限于其中某一种基因并且研究都是针对美洲及非裔美洲人种。对于亚洲人种(中国)这方面的研究暂未见报道。
目的
本研究将立足早产胎膜早破为契机,从分子及蛋白水平上研究PPROM与其胎膜组织上的基质金属蛋白酶表达量之间相关性并进行验证,同时在基因水平上进一步研究PPROM与基质金属蛋白酶基因多态性之间相关性,并结合PPROM相关高危因素共同分析三者相互关系,探讨基质金属蛋白酶基因多态性与母体因素共同作用对PPROM易感性影响,旨在为早期预测早期预防PPROM提供科研依据,提高母婴生存率及生存质量有重大临床意义。
方法
一、早产胎膜早破相关高危因素分析
选择2009年5月至2010年1月,在广州、深圳市三甲(广州南方医科大学附属南方医院、广州医学院附属三院、深圳市妇婴医院、深圳市罗湖人民医院)及二甲医院(南方医科大学附属三院华瑞医院、广州武警总队医院番禺分院、深圳市西丽人民医院)住院分娩的早产胎膜早破的孕妇共78例,足月胎膜早破的孕妇107例,自发性早产的孕妇59例,各组均在同一时期按一定比例在各家医院抽取,同时随机抽取同时期入院分娩的正常足月产的孕妇171例。对每一位研究对象入院后进行病史询问,并记录孕妇年龄、体重、户籍情况、文化水平、职业、健康情况、系统产检与否。并询问是否既往早产或早产胎膜早破史、孕期阴道流血史、孕期生殖道炎症史、宫颈机能不全或宫颈管≤30mm、绒毛膜羊膜炎、吸烟或被动吸烟、羊水过多、双胎妊娠、人工流产次数、孕期羊膜腔穿刺术或胎儿镜检查、孕期额外补充维生素等。临床病史资料由各位孕妇管床医师协助完成询问并记录,并经过患者知情同意。原始病历资料统一输入EXCEL电子表格之后进行统计学分析。
二、胎膜组织中MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9)表达差异的研究
1、采集研究对象外周静脉血提取DNA,应用荧光定量PCR反应(SYBR Green法Real-time PCR)方法分析MMPs mRNA含量：荧光定量仪ABI 7500全自动荧光定量PCR仪检测反应结束后,由电脑自动分析并计算结果。结果按拷贝数分析,用B表示,即B=拷贝数／μl cDNA。考虑到各个样本总RNA浓度的差异,最终计算结果按下列公式换算：A=B1(目的基因)÷B2(内参基因)。
2、采集研究对象胎膜组织提取RNA,应用Western-blot法分析胎膜组织上MMPs蛋白表达量：以GAPDH为内参,每组均相同条件重复3次,待膜干后对其进行扫描保存,图像处理系统分析显色条带灰度值以判断蛋白含量高低。各组MMPs在胎膜组织中均有不同程度表达并进一步验证了Real-time PCR法定量的结果。
三、早产胎膜早破与基质金属蛋白酶基因多态性及母体因素关联性研究
应用基质辅助激光解吸附电离飞行时间质谱法(MALDI-TOF)检测MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9)的多个位点多态性之后,由各组质谱图的信号峰可看出MMPs各SNP位点中：MMP-8(rs1320632、rs2155052),MMP-9 (rs3918242)仅出现一个T基因型信号峰,未出现多态性表现,因此剔除。余下各位点分析其多态性即SNP基因型在病例对照组中的分布是否符合Hardy—Weinberg平衡并计算基因型、等位基因型分布频率及其结合母体因素分析与PPROM的关联性。
四、统计学处理
采用SPSS16.0进行统计学分析。计量资料统计学描述采用x±s,计数资料采用频数和百分比表示。计量资料两组组间差别比较采用成组t检验,若方差不齐采用Mann-Whitney U检验；多组组间差异比较采用方差分析,组间两两比较采用LSD-t检验,若方差不齐,采用Kruskal-Wallis H检验,组间两两比较采用Mann-Whitney U检验,多重检验校正采用Bonferroni校正。计数资料的组间差异比较采用χ2检验、两两比较采用分割χ2检验、单因素和多因素Logistic回归分析。采用陈大方,陈常中专著中SAS宏程序进行Hard-weinberg遗传平衡检验,并计算基因型、等位基因型分布频率。采用χ2检验、两两比较采用分割χ2检验、单因素和多因素Logistic回归分析进行组间基因型、等位基因分布差异检验。P<0.05认为差异具有统计学意义。
结果
一、早产胎膜早破相关高危因素分析
1、研究人群年龄,孕前体重指数及孕期增重情况：
早产胎膜早破组年龄与各组年龄比较,足月胎膜早破组年龄大于早产胎膜早破组,两者之间差异有统计学意义(P=0.003)；早产胎膜早破组孕期增重小于正常足月产组,它们之间差异有统计学意义(P<0.001)。
2、研究人群一般临床资料情况分析：
早产胎膜早破组与足月胎膜早破组比较,绒毛膜羊膜炎在两组的分布情况的差异存在统计学意义(P=0.030)；早产胎膜早破组与正常足月产组比较,系统产检、既往早产或早产胎膜早破史、孕期阴道流血史、绒毛膜羊膜炎、吸烟或被动吸烟、双胎妊娠、体外受精胚胎移植术在两组分布情况的差异存在统计学意义。(P<0.05)。
3、早产胎膜早破相关危险因素的单因素Logistic回归分析：
以早产胎膜早破为病例组,以自发性早产组、足月胎膜早破组及正常足月产组为对照组,分析与早产胎膜早破有关联的高危因素。
早产胎膜早破组与足月胎膜早破组比较,年龄是危险因素(OR=0.921,95%CI：0.872-0.973)；早产胎膜早破组与足月正常产组比较,与早产胎膜早破相关的保护因素有：孕期增重(OR=0.707,95%CI：0.623-0.803)、系统产检(OR=0.506,95%CI：0.294-0.871)、人工流产次数少于1次(OR=0.576,95%CI：0.334-0.994)；相关的危险因素有：既往早产或早产胎膜早破史(OR=9.189,95%CI:1.010-83.619)、孕期阴道流血史(OR=5.125,95%CI:1.978-13.280)、绒毛膜羊膜炎(OR=4.64,95%CI:1.951-11.060)、吸烟或被动吸烟(OR=2.174,95%CI：1.253-3.771)。
二、胎膜组织中MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9)表达差异
早产胎膜早破组与足月胎膜早破组相比较,MMP2, MMP7表达降低,MMP3, MMP8表达增高,且它们之间差异存在统计学意义(P<0.001), MMP1, MMP9表达无明显统计学差异；与自发性早产组比较,MMP2, MMP7表达增高,MMP8表达降低,且它们之间差异存在统计学意义(P<0.001), MMP1, MMP3, MMP9表达无明显统计学差异；而早产胎膜早破组与正常足月产组比较,MMP1, MMP2, MMP3, MMP7, MMP9表达增高,且它们之间差异存在统计学意义(P<0.001),MMP8表达无明显统计学差异。Western-blot进一步验证其蛋白表达量的差异：MMP-1在早产胎膜早破组、自发性早产组、足月胎膜早破组的表达高于正常足月产组；MMP-2在早产胎膜早破组、足月胎膜早破组的表达高于自发性早产组和正常足月产组,足月胎膜早破组的表达高于早产胎膜早破组；MMP-3在早产胎膜早破组、自发性早产组的表达高于足月胎膜早破组和正常足月产组；MMP-7在足月胎膜早破组的表达高于早产胎膜早破组、自发性早产组和正常足月产组；MMP-8在自发性早产组的表达高于早产胎膜早破组、足月胎膜早破组和正常足月产组；MMP-9在正常足月产组的表达低于自发性早产组、早产胎膜早破组和足月胎膜早破组。
三、早产胎膜早破与基质金属蛋白酶基因多态性及母体因素关联性：
1、Hardy—Weinberg平衡检验病例对照组基因型：本研究采用SAS9.1统计学软件对正常足月产组进行Hardy—Weinberg平衡检验,除MMP-2 (rs243866) MMP-8 (rs11225395)外(P=0.049,P=0.014),其余各MMPs-SNP位点基因型频率均通过平衡检验(P>0.05),说明本研究收集的样本基本具有一定人群的代表性,适合做候选易感基因研究。
2、早产胎膜早破组与自发性早产组、足月胎膜早破组及足月正常产组等各对照组MMPs基因SNPs基因型分布频率、等位基因分布频率的比较
(1)早产胎膜早破组与早产组、足月胎膜早破组及足月正常产组等各对照组MMPs基因型分布频率比较结果：
早产胎膜早破组分别与足月胎膜早破组、自发性早产组及正常足月产组比较,早产胎膜早破组与足月胎膜早破组相比,MMP1 (rs1799750)、MMP3 (rs61781、rs35068180)各基因型分布频率存在差异具有统计学意义(P<0.01)；与自发性早产组相比,MMP2 (rs243865、rs243866、rs2285053、rs17859821)、MMP3 (rs35068180)各基因型分布频率存在差异具有统计学意义(P<0.01)；与正常足月产组相比,MMP1 (rs1799750)、MMP2 (rs2285053、rs17859821)、MMP3 (rs35068180)、MMP8 (rs11225395)各基因型分布频率存在差异具有统计学意义(P<0.01)。
(2)早产胎膜早破组与自发性早产组、足月胎膜早破组及足月正常产组等各对照组MMPs等位基因分布频率比较结果：
早产胎膜早破组分别与足月胎膜早破组、自发性早产组及正常足月产组比较,早产胎膜早破组与足月胎膜早破组相比,MMP3 (rs617819、rs35068180)、各等位基因分布频率存在差异,具有统计学意义(P<0.05)；与自发性早产组相比,MMP2 (rs243865、rs243866)、MMP3 (rs35068180)各等位基因分布频率存在差异,具有统计学意义(P<0.05)；与正常足月产组相比,MMP2 (rs2285052、rs2285053)、MMP3 (rs35068180)各等位基因分布频率存在差异具有统计学意义(P<0.01)。
3、早产胎膜早破相关危险因素Logistic回归分析：
Ⅰ、单因素分析：
1、MMPs各基因型分布频率与早产胎膜早破关联性分析：
早产胎膜早破组MMP1 (rs1799750) G.DEL基因型分布频率与GG基因型分布频率比较,G.DEL基因型分布频率高于正常足月产组,且差异有统计学意义,携带G.DEL基因型会增加早产胎膜早破的风险(OR=2.402,95%CI：1.336-4.320). MMP2(rs2285052)CA基因型分布频率与CC基因型分布频率比较,CA基因型分布频率低于正常足月产组,且差异有统计学意义,携带CA基因型降低早产胎膜早破的风险(OR=O.140,95%CI：0.032-0.606)。MMP2(rs2285053) CT基因型分布频率与CC基因型分布频率比较,CT基因型分布频率高于正常足月产组,且差异有统计学意义,携带CT基因型会增加早产胎膜早破的风险(OR=3.200,95%CI：1.828—5.603)。
2、MMPs各等位基因分布频率与早产胎膜早破关联性分析：
早产胎膜早破组与正常足月产组MMP2(rs243865)T等位基因分布频率与C等位基因分布频率比较,携带T等位基因降低早产胎膜早破的风险,是发生早产胎膜早破的保护因素(OR=0.498,95%CI：0.257-0.966)。早产胎膜早破组与正常足月产组MMP2(rs243866)G等位基因分布频率与A等位基因分布频率比较,携带G等位基因使早产胎膜早破的风险增高(OR=2.007,95%CI：1.035-3.891)。早产胎膜早破组与正常足月产组MMP2(rs2285052)A等位基因分布频率与C等位基因分布频率比较,携带A等位基因是发生早产胎膜早破的保护因素,降低早产胎膜早破的风险,(OR=0.152,95%CI：0.036-0.645)。早产胎膜早破组与正常足月产组MMP2(rs2285053)T等位基因分布频率与C等位基因分布频率比较,T等位基因分布频率高于正常足月产组,且差异有统计学意义,携带T等位基因使早产胎膜早破的风险增高(OR=1.795,95%CI：1.155-2.791,)。
Ⅱ、多因素分析：
1、MMPs基因型结合母体因素等多因素分析：
早产胎膜早破组与正常足月产组MMPs基因型结合母体因素等因素进行多因素Logistic回归分析比较发现,早产胎膜早破的危险因素有：①有孕期阴道流血史(OR=3.844,95%CI：1.233-11.984),②有绒毛膜羊膜炎(OR=2.898,95%CI：1.047-8.022),③有吸烟或被动吸烟史(OR=2.716,95%CI：1.383-5.334),④携带MMP1(rs1799750)GG基因型(OR=2.766,95%CI：1.337-5.724),⑤携带MMP2 (rs2285053) CC基因型(OR=6.482,95%CI：3.166-13.271)；发生早产胎膜早破的保护因素：携带MMP2(rs2285052)CC基因型(OR=0.121,95%CI：0.024-0.608)、孕期有系统产检(OR=0.470,95%CI：0.245-0.903)。
2、MMPs等位基因结合母体因素等多因素分析：
早产胎膜早破组与正常足月产组MMPs等位基因结合母体因素等多因素进行多因素Logistic回归分析比较发现,早产胎膜早破的危险因素有：有既往早产或早产胎膜早破史(OR=8.476,95%CI：1.139-63.094),有孕期阴道流血史(OR=6.759,95%CI：2.72-16.759),有绒毛膜羊膜炎(OR=3.424,95%CI：1.508-7.771),有吸烟或被动吸烟史(OR=2.483,95%CI：1.74-4.183),携带MMP8(rs11225395)A等位基因(OR=4.753,95%CI：2.643-8.547)；发生早产胎膜早破的保护因素：携带MMP2(rs243866)A等位基因(OR=0.071,95%CI：0.023-0.222),携带MMP2(rs2285053)C等位基因(OR=0.031,95%CI：0.012-0.082),携带MMP3(rs617819)C等位基因(OR=0.410,95%CI：0.212-0.793),孕期有系统产检(OR=0.537,95%CI：0.324-0.892)。
结论
一、与早产胎膜早破有关的保护因素有：孕期增重、系统产检、人工流产次数少于1次；相关的危险因素有：既往早产或早产胎膜早破史、孕期阴道流血史、绒毛膜羊膜炎、吸烟或被动吸烟。
二、早产胎膜早破组与正常足月产组比较,早产胎膜早破组MMP1, MMP2, MMP3, MMP7, MMP9表达增高,而两组MMP8表达无明显统计学差异。
三、MMP1(rs1799750)G.DEL基因型、MMP2(rs2285053)CT基因型、MMP2(rs2285052)CA基因型与早产胎膜早破发生存在关联性。MMP2(rs243865)T等位基因、MMP2(rs2285052)A等位基因、携带MMP2(rs243866)G等位基因、MMP2(rs2285053)T等位基因与早产胎膜早破发生也存在关联性。四、有孕期阴道流血史,有绒毛膜羊膜炎,有吸烟或被动吸烟史等外界母体因素均能影响MMP1(rs1799750)GG基因型,MMP2(rs2285053)CC基因型,它们均是危险因素；而早产胎膜早破的保护因素有：携带MMP2(rs2285052)CC基因型、孕期有系统产检；
五、有既往早产或早产胎膜早破史,有孕期阴道流血史,有绒毛膜羊膜炎,有吸烟或被动吸烟史等母体因素均能影响MMP8(rs11225395)A等位基因,且均为早产胎膜早破的危险因素；而早产胎膜早破的保护因素有：携带MMP2(rs243866)A等位基因,MMP2(rs2285053)C等位基因,MMP3(rs617819)C等位基因,孕期有系统产检；
Premature rupture of membranes (preterm premature rupture of membrance, PPROM) is one of the key factors leading to preterm delivery, accounting for 25% of preterm birth. Moreover, approximately 30-40% of preterm birth is related with PPROM. Of which before 36 weeks of gestation PPROM will appear the more serious complications of pregnancy, the incidence increasing trend in recent years. Preterm premature rupture of membranes accounted for the reasons as 1/3 of obstetric complications, early prediction of major clinical significance of early prevention, to some extent reduce the preterm birth rate, and improve maternal and newborn survival.
For a long time, premature rupture of membranes is a potenially crisis before onset of premature labor, which is often combinated with it. Furthermore, premature labor become inevitable in preterm premature rupture of membranes, all both of each other. Therefore, it is particularly important to analysis of risk factors on the PPROM and its early diagnosis, as well as the early preventional treatment, which plays a significantly role in clinical The previous studies indicate that matrix metalloproteinases (MMPs) belong to endogenous enzyme which could degrade all protein components in extracellular matrix and the corresponding components of extracellular matrix. It also can control content and fetal membrane extracellular matrix structure, causing weakening of the structure of fetal membranes, leading to premature rupture of membranes. With further study, the etiology of premature rupture of membranes has step in the genomic era. As polymorphisms of MMPs gene are associated with PPROM between the mother and the fetus, pregnant women who carry susceptibility genes also increased the risk of occurrence of PPROM. However, susceptibility genes is characterized by genetic mutations itself does not directly lead to diseases, but a potential risk of the disease increased. That is to say diseases are occurred when the external harmful factors are involved in. The reason why different susceptibility, resistance and other biological characteristics of diseases between different groups and individuals are based on the variation of human genome DNA sequence, and single nucleotide polymorphism (SNP) is the most common one. Recently, the studies show that fetal MMP-1, MMP-9 and MMP-8 polymorphisms among MMP family in certain gene promoter region are related with PPROM.
However, at present the research on SNP screening in MMPs which is related to mechanisms of PPROM and maternal gene polymorphism analysis are poorly known. Furthermore, it is limited to a particular gene and the study always aim at the Americas and African-American race. While asian ethnic origin (China) research in this area has not yet reported.
Objective
this study is in based on preterm premature rupture of membranes, from the molecular and protein level to verify correlation between of MMP expression on fetal membranes and PPROM, then further studies at the genetic level, to verify correlation between of MMPs gene polymorphism and PPROM and to analysis the revelance of them by combinning with maternal factors.at last,we analysis the relationship of the affect of MMPs gene polymorphism and maternal factors and it's common affect to the susceptibility of PPROM.my objective is provide scientific basis for early prevention and early prediction of PPROM.It will be profit for improving maternal and child survival rate and improving quality of life.It is a major clinical significance work.
Methods
一、Analysis of risk factors related to premature rupture of membranes
Select the cases of premature rupture of membranes group of pregnant women, premature rupture of membranes in pregnant women, pregnant women in spontaneous preterm labor and normal term natural delivery pregnant women as a case-control group in seven Guangdong hospitals delivery from May 2009 to January 2010.
二、Fetal membranes MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) expression Differences
1, collection of peripheral blood of objects to extract DNA, application of quantitative PCR reaction (SYBR Green method Real-time PCR) method to analysis MMPs mRNA content.
2, collection of fetal membranes of objects to extract RNA, application of Western-blot analysis protein expression of MMPs on fetal membranes.
三、study the relevance of premature rupture of membranes and matrix metalloproteinase gene polymorphisms and environmental factors
Application of matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) detection of MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) polymorphic locus and then analysis the genotype distribution of case-control group by Hardy Weinberg, calculate the genotype, allele frequency distribution and then analysis the relevance of PPROM.in that and the combination of environmental factors.
四、Statistical analysis
By SPSS16.0 for statistical analysis. Statistical description of measurement data used x±s, Statistical description of count data used frequency and percentage. The differences between the two groups of measurement data used to compare the group t test, if the missing variance with Mann-Whitney U test; multiple groups were compared with analysis of variance compared between groups using LSD-t test, if uneven variance using the Kruskal-Wallis H test was used to compare two groups, Mann-Whitney U test, multiple testing correction using Bonferroni correction. Count data was used to compare differences between groups tested, compared with the separate tests, univariate and multivariate Logistic regression analysis. P<0.05 considered statistically significant difference. Generous with Chen Da Fang,Chen Chang Zhong secondary procedures with the SAS macro genetic Hard-weinberg equilibrium test, and calculate genotype and allele frequency distribution. Use of test used to compare with the separate tests, univariate and multivariate Logistic regression analysis group genotype and allele distribution between tests. P<0.05 considered statistically significant difference.
Result
一、Analysis of risk factors related to premature rupture of membranes
1、study population age, pre-pregnancy body mass index and weight gain during pregnancy situation.
The age of premature rupture of membranes group is larger than premature rupture of membranes group, there was significant difference between the two (P= 0.003); weight in duration of pregnancy of premature rupture of membranes is less than the normal full-term group, there was statistically significant differences between them (P<0.001).
2、Analysis of clinical data in the general population:
Premature rupture of membranes group compared with the premature rupture of membranes, chorioamnionitis, it's distribution of the two groups, there were statistically significant differences (P=0.030); premature rupture of membranes group and the normal full-term group comparison, there were statistically significant differences in two group of medical examination of duration of pregnancy, past history of preterm or premature rupture of membranes, history of vaginal bleeding during pregnancy chorioamnionitis, smoking or passive smoking, twin pregnancy, in vitro fertilization and embryo transplantation in treatment. (P<0.05).
3、analysis the risk factors related to premature rupture of membranes by Logistic regression analysis of single factor:
age is a risk factor for remature rupture of membranes group and premature rupture of membranes group, premature rupture of membranes group compared with term normal production group, the protective factors are:weight gain during pregnancy, medical examination of duration of pregnancy, the number of abortions less than two times;the related risk factors are:previous history of preterm or premature rupture of membranes, history of vaginal bleeding during pregnancy,chorioamnionitis, smoking or passive smoking.
二、MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) expression difference in fetal membranes.
Premature rupture of membranes group compared with the premature rupture of membranes group, MMP2, MMP7 expression decreased, MMP3, MMP8 expression increased, and there were statistically significant differences between them (P <0.001), MMP1, MMP9 expression was not significantly significant difference; with spontaneous preterm birth group, MMP2, MMP7 expression increased, MMP8 expression decreased, and there were statistically significant differences between them (P<0.001), MMP1, MMP3, MMP9 expression was not significant statistical difference; and premature premature rupture of membranes group compared with the normal full-term, MMP1, MMP2, MMP3, MMP7,MMP9 expression increased,and there were statistically significant differences between them (P<0.001), MMP8 there was no significant statistical difference. Western-blot to further verify the differences in protein expression:MMP-1 in the group of premature rupture of membranes, spontaneous preterm birth group, premature rupture of membranes group was higher than the normal full-term group; MMP-2 in premature premature rupture of membranes group, premature rupture of membranes group was higher than spontaneous preterm group and the normal full-term group, premature rupture of membranes group was higher than premature rupture of membranes group; MMP-3 in premature rupture of membranes group, spontaneous preterm premature rupture of membranes group was higher than the normal full-term group and the group; MMP-7 expression in premature rupture of membranes group than premature rupture of membranes group, spontaneous preterm labor group and the normal full-term group; MMP-8 expression in the spontaneous preterm group than premature rupture of membranes group, premature rupture of membranes group and the normal full-term group; MMP-9 in the normal full-term group was less than spontaneous preterm birth group, premature rupture of membranes group and premature rupture of membranes group.
三、relevance of premature rupture of membranes and matrix metalloproteinase gene polymorphisms associated with maternal factors.
1, use Hardy Weinberg equilibrium to test the genotype of case-control group:This study used statistical software SAS9.1 to to carry out Hardy Weinberg equilibrium test for normal full-term group. in addition to MMP-2 (rs243866) MMP-8 (rs11225395), the genotypes all passed the balancing test in the remaining MMPs-SNP locus, it shows the collected samples in this study is the basic representation of certain groups, it suitable to become the candidate susceptibility genes.
2, genotype frequency of MMPs gene SNPs, allele frequency comparison in spontaneous preterm group,premature rupture of membranes group, premature rupture of membranes group and full-term normal control group.
(1) genotype frequency comparison MMPs in preterm premature rupture of membranes group, spontaneous preterm group, premature rupture of membranes group and normal control group.
Premature rupture of membranes group compared with the premature rupture of membranes group, there were statistically significant differences in that genotype frequencies of MMP1 (rs1799750), MMP3 (rs617819, rs35068180); compared with spontaneous preterm birth group, there were statistically significant differences in that genotype frequencies of MMP2 (rs243865, rs243866, rs2285053, rs17859821), MMP3 (rs35068180); compared with the normal full-term, there were statistically significant differences in that genotype frequencies of MMP1 (rs1799750), MMP2 (rs2285053, rs17859821), MMP3 (rs617819, rs35068180), MMP8 (rs11225395).
(2) allele frequency comparisons MMPs in preterm premature rupture of membranes group, spontaneous preterm group, premature rupture of membranes group and normal control group.
Premature rupture of membranes group compared with the premature rupture of membranes group, there were statistically significant differences in that allele frequency of MMP3 (rs617819, rs35068180); compared with spontaneous preterm group, there were statistically significant differences in that allele frequency of MMP2 (rs243865, rs243866), MMP3 (rs35068180); compared with the normal full-term group, there were statistically significant differences in that allele frequency of MMP2 (rs2285052, rs2285053), MMP3 (rs35068180).
3、relevance of risk factors of premature rupture of membranes by Logistic regression analysis:
Ⅰ, single-factor analysis
(1) Analysis the relevance of MMPs frequency distribution of the genotypes associated with premature rupture of membranes
MMP1 (rs 1799750) G. DEL genotype frequency in premature rupture of membranes group is higher than that is in the normal full-term group, the difference was statistically significant, carry G. DEL genotype will increase risk of premature rupture of membranes. MMP2 (rs2285052) CA genotype frequencies lower than the normal full-term group, the difference was statistically significant, carrying CA genotype does not increase the risk of premature rupture of membranes. MMP2 (rs2285053) CT genotype frequency was higher than the normal full-term group, the difference was statistically significant, carry CT genotype will increases the risk of premature rupture of membranes.
(2) Analysis the relevance of MMPs frequencies distribution of the allele associated with premature rupture of membranes
Premature rupture of membranes group compared with normal full-term group, carrying MMP2 (rs243865) T allele does not increase the risk of premature rupture of membranes, it is the protective factors for premature rupture of membranes.Premature rupture of membranes group compared with normal full-term group, compared with MMP2 (rs243866) G allele carried an increased risk of preterm premature rupture of membranes (OR=2.007,95% CI:1.035-3.891, P= 0.039). preterm premature rupture of membranes group compared with normal full-term group, MMP2 (rs2285052) A allele is the protective factor for preterm premature rupture of membranes, does not increase the risk of premature rupture of membranes. preterm premature rupture of membranes group compared with normal full-term group, it carrying with MMP2 (rs2285053) T allele frequency was higher than the normal full-term group, the difference was statistically significant, increased risk of preterm premature rupture of membranes.Ⅱ, multi-factor analysis
(1) MMPs genotype combination of maternal factors
analysised the factor of MMPs genotype combination of maternal factors in preterm premature rupture of membranes group and normal full-term group by used multi-factor multi-factor comparison Logistic regression analysis, such as showed that risk factors for preterm premature rupture of membranes were:a history of vaginal bleeding during pregnancy, there chorioamnionitis, history of smoking or passive smoking, carrying MMP1 (rs1799750) GG genotype, carrying MMP2 (rs2285053) CC genotype; while carrying MMP2 (rs2285052) CC genotype, medical examination of duration of pregnancy are likely the protective factor for preterm premature rupture of membranes.
(2) MMPs allele combination of maternal factors
analysised the factor of MMPs allele combination of maternal factors in preterm premature rupture of membranes group and normal full-term group by used multi-factor multi-factor comparison Logistic regression analysis, such as showed that risk factors for preterm premature rupture of membranes were:previous history of premature delivery or premature rupture of membranes, the history of vaginal bleeding during pregnancy, chorioamnionitis, history of smoking or passive smoking,, carrying MMP8 (rs11225395) A allele; carrying MMP2 (rs243866) A allele, carrying MMP2 (rs2285053) C allele, carrying MMP3 (rs617819) C allele, medical examination of duration of pregnancy may be a protective factors for preterm premature rupture of membranes.
Conclusion
1、the protective factors related to preterm premature rupture of membranes are: weight gain during pregnancy, medical examination of duration of pregnancy, abortion times≤1, the risk factors related to preterm premature rupture of membranes are past history of preterm or premature rupture of membranes, vaginal bleeding during pregnancy history of chorioamnionitis, smoking or passive smoking,.
2、preterm premature rupture of membranes group compared with normal full-term group, the expression of MMP1, MMP2, MMP3, MMP7,MMP9 in preterm premature rupture of membranes group is increased, there was no significant statistical difference in the expression of MMP8 in that.
3, there is relevance in preterm premature rupture of membranes with MMP1 (rs1799750) G. DEL genotype, MMP2 (rs2285053) CT genotype, MMP2 (rs2285052) CA genotype. there is relevance in preterm premature rupture of membranes with MMP2 (rs243865) T allele, MMP2 (rs2285052) A allele, carrying MMP2 (rs243866) G allele, MMP2 (rs2285053) T allele.
4, MMP1 (rs1799750) GG genotype, MMP2 (rs2285053) CC genotype were affect-ed maternal factors,such as:history of vaginal bleeding during pregnancy, history of chorioamnionitis, history of smoking or passive smoking.they were risk factors and protective factors of premature rupture of membranes is include:carrying MMP2 (rs2285052) CC genotype, medical examination of duration of pregnancy;
5, MMP8 (rs11225395) A allele were affect-ed maternal factors,such as:past history of preterm labor or premature rupture of membranes, history of vaginal bleeding during pregnancy, chorioamnionitis, history of smoking or passive smoking.they were risk factors for preterm premature rupture of membranes; and protective factors of premature rupture of membranes is include:carrying MMP2 (rs243866) A allele, MMP2 (rs2285053) C allele, MMP3 (rs617819) C allele, medical examination of duration of pregnancy;
引文
[1]Dodd JM,Flenady VJ,Cincolla R,et al.Progesterone for the prevention of reterm birth a systematic review.[J].Obstet Gynecol,2008,112(1):127-134.
[2]杨慧霞,董锐.制定<早产的临床诊断与治疗推荐指南(草案)>的相关说明[J]。中华妇产科杂志,2007,42(7)：433.
[3]杨慧霞,丁依玲,樊尚荣等.早产领域的相关问题[J].现代妇产科进展,2006,16(2)：81-90.
[4]Meis PJ,Klehanoff M,Thom E,et al.Prevention of recurrent preterm delivey by 17 Ipha-hydroxyprogesterone caproate[J].N Engl J Med,2003,348 (24):2379-2385.
[5]Swamy GK,Skjaerven,R.Association of pretermbirth with long-term survival,reproduction,and next-generation preterm birth[J].JAMA,2008,299 (12):1429-1431.
[6]Verma U,Goharkhay N,Beydoun S.Conservative management of preterm premature rupture ofmembranes between 18 and 23 weeks of gestation2maternaland neonataloutcome[J]. Eur J Obstet Gyne-col Reprod Biol,2006,128 (2):119-124.
[7]Mercer.Brian M. Preterm Premature Rupture of the membranes. Obstet Gynecol,2003,101:178-193.
[8]Maymon E,Romero R,Pacora P,et al.Evidece for the participation of interstitial collgenase(matrix metalloproteinase 1 in preterm rupture of membrances[J].AmJ Obstet Gynecol,2000,183:914-920.
[9]Maymon E,Romero R,Pacora P,et al.Human neutrophil collgenase (matrix metalloproteinase 8 in preterm rupture of membrances,and intrauterine infection[J]. Am J Obstet Gynecol,2000,183:94-99.
[10]Biggio JR,Ramsey PS,Cliver SP,et al.Midtrimester amniotic fluid matrix metalloproteinase-8 (MMP-8)levels above the 90th percentile are a marker for subsequent preterm premature rupture of membrances[J].Am J Obstet Gynecol 2005,192:109-113.
[11]Fortanato SJ,Menon R.Distinct molecular events suggest difference pathways for preterm labor and permature rupture of membranes [J]. Am J Obstet Gynecol,2001,184:1399-1405.
[12]Maymon E,Romero R,Pacora P,et al.A role for the 72 kDa gelatinase (MMP-2)and its inhibitor(TIMP-2)in human parturition,premature rupture of membranes and intrauterine infection[J].Perinat Med,2001,29:308-316.
[13]Yonemoto H,Young CB,Ross JT,et al.Changes in matrix metalloproteinase (MMP-2 and MMP-9)in the fetal amnion and chorion during gestation and at term and preterm labor[J].Placenta,2006,27:669-667.
[14]Vadillo OF,Sandowsky DW, Haluska GJ. Identification of matrix metalloproteinase-9 in amniotic fluid and amniochorion spontaneous labor and after experimental intrauterine infection or interleukin-βinfusion in pregnant rhesus monkeys[J]. Am J Obstet Gynecol,2002,186(1):128-138.
[15]Locksmith GJ,Clark P,Duff P.Amniotic fluid concentrations of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 during pregnancy and labor[J]. Am J Obstet Gynecol,2001,184(2):159-164.
[16]Park KH,Chaiworapongsa T,Kim YM,et al.Matrix metalloproteinase 3 in parturition, premature rupture of membranes,and microbial invasion of the amniotic cavity[J].Perinat Med,2003,31(1):12-22.
[17]王红琳,周春,张元珍,基质金属蛋白酶2、9及其特异性组织抑制剂在自发性胎膜早破发病中的作用[J]。中华妇产科杂志2005 Jan;40(1):29-33.
[18]卢丹,王志学,王晓玲等,基质金属蛋白酶9及其组织抑制物1的水平变化 与胎膜早破发病的关系[J]。中华妇产科杂志2006 Jan；41(1)：20-4.
[19]Mingione MJ,Pressman EK,Woods JR..Prevention of PPROM.current and future strategies.[J]Maternal-fetal Neonatal Med,2006,19:783-789.
[20]Maymon E,Romero R,Pacora P,et al.Evidece for the participation of interstitial collgenase(matrix metalloproteinase 1)in preterm rupture of membrances [J].AmJ Obstet Gynecol,2000,183:914-920.
[21]Fujimoto T,ParryS,UrbanekM,et al A single nucleotide polymorphism in the matrix metalloproteinase-1(MMP-1) promoter influences amnion cell MMP-1 expression and risk for preterm premature rupture of the fetal membranes.[J]Biol Chem,2002,277:6296-6302.
[22]Ferrand PE,Samuel PS,SammeM,et al A polymorphism in preterm premature rupture of membrances in African Americans.[J] Mol Human Rep rod,2002,8:494-501.
[23]Pemi SC,Vardhana Skalish R,et al Clara cell protein 16 concentraton in mid trimester amniotic fluid:association with fetal gender,fetal G>A+38CC16 gene polymorphism and pregnancy outcome.[J]Rep rod Immunol,2005,68:85-90.
[24]MclarenJ,TaylorDJ,Bell SC.Increased concentration of promatrix metallopro-teinase 9 in term fetal memmbrances overlying the cervix before labor [J]. Am J Obstet Gynecol,2000,18(3):409-416.
[25]乐杰主编.妇产科学[M].第五版北京人民出版社,2001；163-165.
[26]Cunningham FC,GantNF,LevenoKJ,et al.21st ed New York McGraw-Hill Con Inc, Williams Obstertrics[M],2001:689-727.
[27]陈乐真.妇产科诊断病理学[M].北京：人民军医出版社,2002：437.
[28]Drakeley AJ,Roberts D,Alfirevic Z.cervical cerclage for prevention of preterm delivery:meta-analysis of randomized trails[J].Gynecol Obstet,2003,102:621-627.
[29]夏晓艳,黄醒华.宫颈机能不全的诊治进展[J].实用妇产科杂志,2005,21(4)：210-212.
[30]Draper DL,Kush M,Dorohoe W,et al.Preterm premature rupture of membranes without labor is not associated with increase levels of matrix metalloproteinase-9 protein[J].Prenat Neonat Med,2001,6:219-226.
[31]刘志杰,刘淑云.核因子NF-κB在早产及分娩发动中的研究[J].国外医学妇幼保健分册,2005,16(6)：358-360.
[32]Tanya M.M,Hill D.A.Preterm premature rupture of membranes:diagnosis and management[J].American family physician,2006,73(4):659-664.
[33]周健,段涛.未足月胎膜早破的病因学研究进展[J].中国实用妇科与产科杂志.2006,22(6)：457-459.
[34]Pararas M.V,Skevaki C.L,Kafetzis D.A.Preterm birth due to maternal infection: causative pathogens and modes of prevention[J].European journal of clinical microbiology&infectious diseases 2006,25:562-569.
[35]曾蔚越.早产与早产儿.[M]第1版北京人民军医出版社,2006；38-39.
[36]Zeitlin JA,Ancel PY,Saurel CMJ,et al..Are risk factor the same for small fou gestational age versus other preterm births?Am J Obstet Gynecol, 2001;185(1):208.
[37]Nien JK,Yoon BH,Espinoza J,et al.A rapid MMP-8 bedside test for the detection of intra-amniotic inflammation identifies patients at riske for imminnent preterm delivery [J].Am J Obstet Gynecol,2006,195(4):1025-1030.
[38]Romoro R, Chainorapongsa T, Espinoza J. Fetal plasma MMP-9concentrations are elevated in preterm premature rupture of the membranes [J]. AmJ Obstet Gynecol,2002,187:1125～1130.
[39]Weiss A, Goldman S, Shalev E.. The matrix metalloproteinases (MMPS) in the decidua and fetal membranes[J]. Front Biosci.2007 Jan 1;12:649-59.
[40]Ekwo, EE, Gosselink, CA, Moawad, A. Unfavorable outcome in penultimate pregnancy and premature rupture of membranes in successive pregnancy[J]. Obstet Gynecol 1992; 80:166.
[41]CollinsFS,BrooksLD,AhakravartiA,et al.DNA polymorphism discovery resource for research on human genetic variation[J]. Genome Res, 1998,8:12291.
[42]StephensM, SmithNJ, DonnellyP. A new statistical method for haplotype pedigree on structure from populationdata.AmJHumGenet.2001,68:978-989.
[43]顾明亮,褚嘉佑.基于标签单核昔酸多态性单倍型和单倍域的构建及其在关联研究中的应用[J].中华医学遗传学杂志,2007,24：660-665.
[44]Karas M,Hillenkamp F.Laser desorption Ionization of proteins with mloecular masses exceeding 10000 Daltons[J].Anal chen,1988,60:2299-2301.
[45]Haff L,Smirnov I.Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry[J].Genome Res,1997,7:378-388.
[46]Ross P,Hall L,Smirnov I,Haff L.High level multiplex genotyping by MALDI-TF mass spectrometry[J].Nat Blotechnol,1998,16:1347-1351.
[47]Braun A,Little D P.,Koster H.Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry[J].Clin Chem,1997,43:1151-1158.
[48]Tang K,Fu D J,Julien D,Braun A,et al,Chip-base genotyping by mass spectrometry[J].Proc Natl Acad Sci USA,1999,96:10016-10020.
[49]Nelson M R,Mamellos G,Kammerer S et al.Large-scale validation of single nucleotide polymorphisms in gene regions[J]. Genome Res,2004,14: 1664-1668.
[50]Ross PL,Lee K,Belgrader P et al.Discrimination of single—nucleotide polymorphisms in human DNA using peptide nucleic acid probes detected by MALDI-TOF mass spectrometry[J]. Anal Chem,1997,69:4197-4202.
[51]Ye S,Liang X,Yamamoto Y et al.Detection of single nucleotide polymorphisms by the combination of nuclease S1 and PNA[J].Nucleic Acids Res,2002,Suppl 235-236.
[52]Griffin TJ,Hall JG,Prudent JR,et al.Direct genetic analysis by matrix-assisted laser desorption ionization mass spectrometry[J].Proc Natl Acad Sci USA,1999,96:6301-6306.
[53]Nishioka Y,Sagae S,Nishikawa A,et al.A relationship between Matrix metalloproteinase-1(MMP-1)promoter polymorphism and cervical cance progression[J].Cancer Lett,2003,200:49-55.
[54]Ye S,Eriksson P,Hamsten A,et al.Progression of coronary atherosclerosis are associated with a common genetic variant of the human stromelysis-1 promoter which results in reduced gene expression[J]. Biol Chem,1996, 271:13055-1306.
[55]WangH,Parry S,MaconesG,et al.Functionally significant SNPMMP8 promoter haplotypes and pretern premature ruputre of membranes(PPROM)[J].Hum MolGenet2004,1;13(21):2659—2669.
[56]Perni SC, Vardhana S, Kalish R, et al. Clara cell protein 16 concentration in mid-trimester amniotic fluid:association with fetal gender, fetal G>A + 38 CC16 gene polymorphism and pregnancyoutcome[J]. J Reprod Immunol, 2005,68(1-2):8590.
[57]Jormsjo S,Whatling C,WalterDH,et al.Allele specific reuglation of matrix metalloproteinase-7 promoter activiyt is assoeiated with coronary artey luminal dimensions among hypercholesterolemic patients[J]. ArteriosclerThrombVasc Biol,2001,21:1834-1839.
[1]郭海英,马秀菊。胎膜早破的发病因素和发病机制的研究进展[J]。中国妇幼健康研究,2007；18(6)：501-504.
[2]乐杰.妇产科科学[M]第六版.北京：人民卫生出版社,2004,145.
[3]Karat C,Madhivanan P,Krupp K,The clinical dand microbiological correlates of premature rupture of membranes [J].Idian J Med Microbiol,2006 Oct,24(4):283-5.
[4]Mingione MJ, Pressman EK, Woods JR. Prevention of PPROM current and future strategies. J Materna-Fetal Neonatal Med,2006,19:7832789.
[5]Kalish RB, Vardhana S, Gupta M, et al. Polymorphisms in the tumor necrosis factor-alpha gene at position2308 and the inducible 70 kd heat shock protein gene at position +1267 in multifetal pregnancies and preterm premature rupture of fetal mem-branes. AmJ Obstet Gynecol,2004,191:136821374.
[6]Matthew JM, Eva K P, James R W, et al. Prevention of pPROM:Current and future strategies [J]. Journal of M aternal Fetal NeonatalMedicine,2006,19 12:7832789.
[7]薛晓萍,薛瑞娟,杨华,基质金属蛋白酶与未足月胎膜早破[J].中国计划生育学杂志.2007；10(144)：634-636.
[8]张莉,杨海澜.基质金属蛋白酶与胎膜早破[J].中国优生与遗传杂志.2008,16(5)：9-11.
[9]Menon R,Fortunato SJ.The role of matrix degrading enzymes and apoptosis in rapture of membranes [J].SCO Gynecol Investing,2004,11:427-437.
[10]Robert V,Hideaki N.Matrix metalloproteinases and tissue inhibitors of metalloproteinases-structure,function,and biochemistry[J]. Circulation Research,2003,92(8):827-839.
[11]Maymon E,Romero R,Pacora P,et al.Evidece for the participation of interstitial collgenase(matrix metalloproteinase-1) in preterm rupture of membrances[J].AmJ Obstet Gynecol,2000,183:914-920.
[12]Maymon E,Romero R,Pacora P,et al.Human neutrophil collgenase (matrix metalloproteinase 8)in preterm rupture of membrances,and intrauterine infection[J]. Am J Obstet Gynecol,2000,183:94-99.
[13]Biggio JR,Ramsey PS,Cliver SP,et al.Midtrimester amniotic fluid matrix metalloproteinase-8 (MMP-8)levels above the 90th percentile are a marker for subsequent preterm premature rupture of membrances[J].Am J Obstet Gynecol 2005,192:109-113.
[14]Fortanato SJ,Menon R.Distinct molecular events suggest difference pathways for preterm labor and permature rupture of membranes[J]. Am J Obstet Gynecol,2001,184:1399-1405.
[15]Maymon E,Romero R,Pacora P,et al.A role for the 72 kDa gelatinase(MMP-2)and its inhibitor(TIMP-2)in human parturition,premature rupture of membranes and intrauterine infection[J].Perinat Med,2001, 29:308-316.
[16]Yonemoto H,Young CB,Ross JT,et al.Changes in matrix metalloproteinase (MMP-2 and MMP-9)in the fetal amnion and chorion during gestation and at term and preterm labor[J].Placenta,2006,27:669-667.
[17]Park KH,Chaiworapongsa T,Kim YM,et al.Matrix metalloproteinase 3 in parturition, premature rupture of membranes,and microbial invasion of the amniotic cavity[J].Perinat Med,2003,31(1):12-22.
[18]王红琳,周春,张元珍,基质金属蛋白酶2、9及其特异性组织抑制剂在自发性胎膜早破发病中的作用[J]。中华妇产科杂志2005 Jan；40(1)：29-33.
[19]卢丹,王志学,王晓玲等,基质金属蛋白酶9及其组织抑制物1的水平变化与胎膜早破发病的关系[J]。中华妇产科杂志2006 Jan；41(1)：20-4.
[20]Fujimoto T,ParryS,UrbanekM,et al A single nucleotide polymorphism in the matrix metalloproteinase-1(MMP-1) promoter influences amnion cell MMP-1 expression and risk for preterm premature rupture of the fetal membranes. [J]Biol Chem,2002,277:6296-6302.
[21]Ferrand PE,Samuel PS,SammeM,et al A polymorphism in preterm premature rupture of membrances in African Americans. [J] Mol Human Rep rod,2002,8:494-501.
[22]Pemi SC,Vardhana Skalish R,et al Clara cell protein 16 concentraton in mid trimester amniotic fluid:association with fetal gender,fetal G>A+38CC16 gene polymorphism and pregnancy outcome.[J]Rep rod Immunol,2005,68:85-90.
